EtxB and CtxB as Carrier Molecules for the A Subunit Escherichia coli (E. coli) heat labile enterotoxin (Etx) and its closely related homologue, cholera toxin (Ctx) from Vibrio cholerae, are examples of protein toxins which bind to glycolipid receptors on host cell surfaces. Each toxin consists of six noncovalently linked polypeptide chains, including a single A subunit (27 kDa) and five identical B subunits (11.6 kDa) which bind to GM-1 ganglioside receptors found on the surfaces of mammalian cells (Nashar et al 1996 Proc Natl Acad Sci 93: 226-230). The A subunit is responsible for toxicity possessing adenosine diphosphate (ADP) ADP-ribosyltransferase activity, whereas the B subunits (EtxB and CtxB) are non-toxic oligomers which bind and cross-link a ubiquitous cell surface glycolipid ganglioside, called GM-1, thus facilitating A subunit entry into the cell.
B Subunit is a Potent Immunogen
In contrast to the poor immunogenicity of the A subunit alone, both EtxB and CtxB are exceptionally potent immunogens and their respective holotoxins, Etx and Ctx (which comprise the A and B subunits) are known to be potent adjuvants when given orally in combination with unrelated antigens (Ruedl et al 1996 Vaccine 14: 792-798; Nashar et al 1993 Vaccine 11: 235; Nashar and Hirst 1995 Vaccine 13: 803; Elson and Ealding 1984 J Immunol 133: 2892; Lycke and Holmgren 1986 Immunology 59: 301). Because of their immunogenicity, both EtxB and CtxB have been used as carriers for other epitopes and antigens (Nashar et al 1993 ibid) and have been used as components of vaccines against cholera and E. coli mediated diarrhoeal diseases (Jetborn et al 1992 Vaccine 10: 130).
B Subunit is a Potent Immunomodulator
We have demonstrated the surprising finding that the EtxB subunit is also capable of acting as an immunomodulator in immune disorders. In this respect, we have disclosed in WO 97/02045 that EtxB binds to GM-1 ganglioside receptors which are found on the surfaces of mammalian cells and that this binding induces differential effects on lymphocyte populations including a specific depletion of CD8+ T cells and an associated activation of B cells.
One of the most unexpected and sting effects of the B-subunits is their capacity to trigger the selective apoptosis of CD8+ T-cells, as well as to alter CD4+ T-cell differentiation, activate B-cells and modulate antigen processing and presentation by macrophages (Williams, N. A., Hirst, T. R. & Nashar, T. O. (1999) Immunol. Today 20, 95-101.). These potent immunological properties have led to testing of the B-subunits as adjuvants for stimulating mucosal and systemic responses to co-administered antigens (Verweij, W. R., de Haan, L., Holtrop, M., Agsteribbe, E., Brands, R., van Scharrenburg, G. J. M. & Wilschut, J. (1998) Vaccine 16, 2069-2076. Richards, C. M., Aman, A. T., T. R., Hill, T. J. & Williams, N. A. (2001) Journal of Virology 75, 1664-1671.); and as agents for down-regulating proinflammatory autoimmune diseases such as rheumatoid arthritis and diabetes (Williams, N. A., Stasiuk, L. M., Nashar, T. O., Richards, C. M., Lang, A. K., Day, M. J. & Hirst, T. R. (1997) Proc. Natl. Acad. Sci. (USA) 94, 5290-5295).
Mutant B Sub-Units—No Gm-1 Binding—No Immunomodulation
These effects are absent when a mutant EtxB protein (G33D) (lacking GM-1 binding activity) is employed. Consequently, these experimental results suggested that all of the functionalities associated with EtxB and CtxB are attributable to the capacity of the EtxB and CtxB subunits to bind to the GM-1 receptor and that immunomodulation and other effects of Etx and Ctx are mediated through GM-1 binding since mutants lacking the capacity to bind GM-1 (such as EtxB (G33D)) fail to act as adjuvants or immunomodulators.
It is well known that CtxB and EtxB contain an extensive conserved segment spanning residues 45 to 74 that contains an exposed loop from Val-52 (V52) to Ile-58 (158) located on the lower convoluted surface of the molecule (Hirst, T. R. (1999) in The Comprehensive Sourcebook of Bacterial Protein Toxins, ed. Freer, J. E. A. a. J. H. (Academic Press, London), pp. 104-129). This loop is normally oriented towards the cell membrane and forms part of the GM1-binding surface, with residues Gln-56, His-57 and Ile-58 involved in a network of solvent-mediated hydrogen bonds that is conserved in the presence of bound GM1-pentasaccharide (Merritt, E. A., Sixma, T. K., Kalk, K. H., Van Zanten, B. A. M. & Hol, W. G. J. (1994) Mol. Microbiol. 13, 745-753.).
Mutant B Sub-Units—Gm-1 Binding—No Immunomodulation
We have demonstrated in WO 00/14114 that CtxB molecules with point mutations at three separate sites within the β4-α2 loop (positions 51, 56 and 57) retained GM-1 binding activity, but lacked other activities, such as toxicity and the capacity to upregulate CD25 and trigger apoptosis of CD8-positive T-cells. We have also shown that EtxB molecules with point mutations in position H57 of EtxB showed a similar loss in triggering/modulation of immune cell populations. In addition, Ctx holotoxins comprising B subunits with mutations also showed a defect in an ability to trigger electrogenic chloride secretion, the primary secretory event responsible for mediating diarrhorea. These findings clearly demonstrated that CtxB and EtxB molecules with point mutations within the β4-α2 loop were capable of binding to the GM-1 receptor but were lacking in an immunomodulatory effect which suggested that not all of the effects of Etx and Ctx and in particular, the immunomodulatory effects, were mediated through but not exclusively by GM-1 binding.
In particular, WO 00/14114 confirmed the importance of the B-subunit E51-I58 loop, and in particular H57 in mediating the immunomodulatory properties of the molecule. The teachings in WO 00/14114 demonstrated that the β4-α2 loop of EtxB/CtxB is responsible for a secondary binding activity and so the use of this loop in isolation from the rest of the EtxB/CtxB molecule (for example as a peptide), may permit the secondary binding activity to occur in the absence of the first. Thus, the selective mutation of the β4-α2 loop, or a peptide derived from this loop, may be exploited with a view to increasing the affinity of the secondary binding activity. By increasing the affinity of the secondary binding activity, the interaction with GM-1 may be further obviated. In summary, the teachings in WO 00/14114 demonstrated that the “secondary” binding activity of an isolated “loop” peptide is not necessarily dependent on a primary GM-1 binding event as is found with full length CtxB and EtxB to mediate the immunomodulatory response.
Thus, it is clear from the above studies that the wild type B subunit is a potent immunogen and a potent immunomodulator whereas the mutations in the B subunit can result in either no GM-1 binding and no immunomodulation or the retention of GM-1 binding but with no immunomodulatory capability.
The Immunological Mechanisms Underlying the Use of the B-Subunit.
The B-subunits ability to modulate the immune response is dependent on its capacity to modulate the activity of T-cells, B-cells and populations of antigen presenting cells. Each of these cell types plays a critical role in the development of the immune response. In the normal response to a foreign organism, antigens are internalised by antigen presenting cells, of which professional antigen presenting cells, such as dendritic cells are the most important. These cells are specialised in breaking down proteins into short amino acid sequences (peptides) which associate with major histocompatibility complex (MHC) molecules which are then transported to the cell surface. Foreign peptides bound to class II MHC molecules are recognised by T-helper cells (CD4+ T-cells) which are activated as a result and begin to divide, differentiate and secrete hormone-like messengers called cytokines. The T-helper cells then co-ordinate and maintain the immune response.
Subsequent responses can involve the activation of i) B-cells which mature into plasma cells capable of producing antibodies, ii) macrophages and neutrophils which enter the sites of infection and ingest foreign material leading to its destruction, and iii) other types of T-cell (CD8+ T-cells) which can recognise virally infected cells of the body and kill them. Most normal immune responses will involve activation of all of these components to some extent. However, it is clear that certain factors can affect which particular components are dominant.
In addition, in certain circumstances it is clearly beneficial to be able to tailor which type of response is elicited. By way of example, it is well known that cytotoxic T lymphocytes (CTLs) play a central role in immune surveillance by recognising foreign antigenic peptides bound to MHC class I molecules and killing virally infected and potentially cancerous cells. Thus, it would be beneficial to tailor the immune response in the direction of the cytotoxic T-cell responses in order to facilitate the removal of infectious agents which reside within cells of the body, such as viruses and certain bacteria.
The effective induction of cytotoxic T-cell responses requires the entry of antigens into the cytosol of antigen presenting cells where they can enter the endogenous class I processing and presentation pathway. However, current immunisation strategies, using peptide or protein antigens, generally fail to elicit a CTL response since these antigens are unable to or are able to only partially access the intracellular compartments where loading of class I molecules occurs. Thus, an efficient delivery system which results in the targeting of antigens into the cytosol is required.
It is known that either wild type EtxB or CtxB may be used as vehicles for the delivery of attached peptides into cells such as MHC Class I bearing cells or professional APCs to achieve the presentation of the such antigenic determinants by MHC class I molecules. The teachings in WO 99/58145 also indicate that the wild type EtxB or CtxB free from of whole toxin, may be used in a conjugate with a peptide or an antigenic determinant to target their delivery to a cell.
One potential disadvantage associated with the use of wild type EtxB or Ctx B is that the potent immune responses engendered to these molecules may preclude their repeated use as drug vehicles. Another potential disadvantage with the use of wild type EtxB or CtxB is that their immunomodulatory capabilities downregulate or suppress certain T-helper responses, that in other circumstances may be beneficial in engendering a preferred or beneficial immune response. Thus, it is desirable to find new ways for delivering an agent to an intracellular compartment of a target cell without triggering a potent immunomodulatory response or a potent immune response such as that induced by wild type CtxB or EtxB molecules.